International Research Journal of Biochemistry and Biotechnology

Vol. 4(1), pp. 068-074, August, 2017. www.premierpublishers.org, ISSN: XXXX-XXXX

IRJBB

Research Article

Epidemiology and Characterization of lipophilic yeast

(Malassezia) from human dandruff samples using
cultural and molecular methods
Sonia Sharma1, Sibi G2*
1
Department of Microbiology, Indian Academy Degree College-Autonomous, Bengaluru, India
2*
Department of Biotechnology, Indian Academy Degree College-Autonomous, Bengaluru, India

Accurate identifications of the species are needed to obtain a better understanding of the role of
each individual species in the etiology of disease. Increased number of species under the genus
Malassezia, urges to characterize the lipophilic yeasts distribution, dominant species from human
dandruff samples. This study aimed at the prevalence of Malassezia species in selected
individuals and their identification using morphological, biochemical methods along with
molecular characterization. Culture based techniques such as catalase test, tween assimilation,
esculin hydrolysis and glycine assimilation were performed and the results revealed that 22
isolates were M. globosa, 12 were M. furfur, 9 were M. sympodialis, 4 were M. obtusa and 3 were
M. restricta out of 50 positive samples. PCR-RFLP method was used for the specific identification
of the Malassezia isolates. 26S rDNA PCR Products after digestion with CfoI and BstF51 revealed
differences between the isolates described the species distribution of Malassezia in dandruff
samples to overcome diagnostic limitations.

Keywords: Malassezia, dandruff, lipophilic, PCR-RFLP, 26S rDNA

INTRODUCTION

Malassezia genus is lipophilic, unipolar budding yeasts,
normally present on the skin of humans and other warm-
blooded vertebrates (Harada et al., 2015; Prohic et al.,
2016). Attention is being paid by dermatologists as
Malassezia can be associated with disease related to
cutaneous and alterations in host defense mechanisms
(Batra et al., 2005; Cafarchia et al., 2004). The yeast
inhabits in sebaceous glands rich sites and has been
implicated in seborrheic dermatitis, folliculitis, dandruff and
atopic dermatitis (Midgley et al., 1998; Gupta et al., 2002).
Fourteen species of Malassezia were described so far
from humans and animals using morphological,
biochemical and molecular characteristics (Hirai et al.,
2004; Sugita et al., 2003, 2004, 2005; Cabenes et al.,
2007; Petry et al., 2011). Malassezia can be differentiated
from other yeast genera based on distinctive
morphological and physiological features but due to the
fact that biochemical and physiological methods have
Epidemiology and Characterization of lipophilic yeast (Malassezia) from human dandruff samples using cultural and molecular methods
some limitations in characterizing closely related
Malassezia species, molecular methods were developed
to the specific identification of the species (Affes et al.,
2009; Gonzalez et al., 2009; Duarte et al., 2010; Park et
al., 2012; Vuran et al., 2014; Ilahi et al., 2017).
Increased number of species under Malassezia has urged
the researchers to know about geographical variation,
species distribution, dominant species and pathogenicity
of this yeast among humans and animals.
*Corresponding author: Sibi G, Department of
Biotechnology, Indian Academy Degree College-
Autonomous, Bengaluru, India. Email address:
gsibii@gmail.com
Sharma and Sibi 069
First stage of this study includes the prevalence of
Malassezia species in selected individuals using
morphological, biochemical methods. In the second stage,
molecular characterization of Malassezia isolates was
performed to overcome the limitations associated with
culture based techniques while characterizing closely
related Malassezia species.
MATERIALS AND METHODS
Collection and Identification of Specimen
Sampling from dandruff patients based on visible flakes
over the scalp was done by scraping the lesions from head
using sterile scalpel and tape method after the institutional
research ethics committee approval. The samples were
stored in sterile containers in refrigerator until used. Direct
microscopy with 20% KOH and methylene blue was
performed and the samples were inoculated on modified
Dixon agar supplemented with tween 80 was used to
cultivate the isolates. After incubation at 37C for 3-5 days,
the colonies were stained with Loefflers methylene blue
for microscopic appearance. Further identification was
done by catalase reaction, tween assimilation and esculin
hydrolysis.
Test organism
Malassezia furfur MTCC 1374 was obtained from Microbial
Type Culture Collection (MTCC), Chandigarh and was
used as reference strain in this study.
Culture Based identification of the isolates
Catalase test
The catalase reaction was determined by application of a
drop of hydrogen peroxide onto a portion of a colony on a
glass slide, or directly on colonies on the culture media.
The production of gas bubbles indicated a positive
reaction.
Tween (20, 40, 60 and 80) utilization tests
For each isolate, the ability to utilize individual Tweens was
tested by the following procedure. Sterile Dixon agar was
melted and allowed to cool to about 50C. The yeast (10 5
cells/ ml) being identified were added (2 mL) to the
medium. The seeded agar was then vigorously mixed and
poured into a petri dish. Once the medium had solidified,
four wells were made by means of a 2 mm diameter punch
and filled with 5 l of 10%, 0.5%, 0.5% and 0.1% of Tween
20, 40, 60 and 80 respectively. The plates were
systematically incubated for one week at 37C. Utilization
of Tweens was assessed by the degree of growth and/or
reaction (precipitation) of the lipophilic yeasts around
individual wells.
Epidemiology and Characterization of lipophilic yeast (Malassezia) from human dandruff samples using cultural and molecular methods
Esculin hydrolysis
The isolates were tested to determine their ability to
metabolize esculin in a nutrient medium. An inoculum from
a pure culture is transferred aseptically to a sterile
petriplate of bile esculin agar and streaked. The inoculated
plates were incubated at 35-37C for 24 hours and the
results were determined. Esculin hydrolysis was observed
if the medium has taken on an intense, chocolate brown
coloration.
Glycine Assimilation
The isolates were inoculated in modified Dixon glycine
media (with 7mM/liter glycine) and the plates were
incubated at 37C for 3 days. Appearance of growth within
2 to 3 days was positive for glycine assimilation.
Molecular Based identification of the isolates
To characterize the isolates of Malassezia spp., culture
based identification such as physiological features and
morphological studies were conducted. Molecular
characterization by PCR-RFLP and the sequencing of the
26S rDNA region in isolate of each species from healthy
individuals and one M. furfur reference strain was
performed.
DNA extraction
Colonies grown on Dixon agar at 32C for 4 to 5 days were
transferred to 500 l of sorbitol citrate buffer (sorbitol 1.1
M, sodium citrate 0.1 M, pH 5.6). To the cell suspension, 1
ml of extracting buffer [Tris-spermidine - 40 mM tris-HCl
(pH 8.0), 4 mM spermidine, 10 mM EDTA, 0.1 M NaCl, 10
mM of -mercaptoetanol, and 0.1% sodium dodecyl
sulfate] supplemented with 50 mg of glucanase was
added. Afterwards, the mixture was incubated at 37C
under agitation in a shaker incubator for 3 h. DNA was
extracted with phenol-chloroform-isoamyl alcohol. In brief,
following a centrifugation at 4C at 14000 rpm for 5 mins,
the aqueous phase was transferred to a new microtube
and the RNA was removed by treatment with 10mg/ml
RNAse for 30min at 37C. After new phenol-chloroform
extraction steps, the DNA was precipitated by addition of
2.5 volumes of ethanol in the presence of 0.3M NaCl
followed by a -20C overnight storage and centrifugation
at 4C at 14000 rpm for 5-10min. The pellet was washed
with 70% ethanol, dried at room temperature, and
suspended in 500 l of TE buffer (pH 8.0) (Sambrook et
al., 1989).
PCR-RFLP of 26S regions
The PCR reaction was performed in a final volume of 50
L. Each reaction contained 1 L of DNA template, 0.5 L
of each primer, 0.20 mm of each deoxynucleoside
triphosphate (dNTPs), 5 L of 10X PCR buffer, and 1.25 U
 Int. Res. J. Biochem. Biotechnol. 070

of Taq polymerase. The primer nucleotides were as
follows: forward, 5TAACAAGGATTCCCCTAGTA-3, and
reverse 5-ATTACGCCAGCATCCTAAG-3. After initial
denaturation at 94C for 5 minutes, the reaction was
followed by 30 cycles of denaturation at 94C for 45
seconds, annealing at 55C for 1 minute, and extension at
72C for 45 seconds, with a final extension step of 72C
for 7 minutes.
bp for M. sympodialis; three bands of ~250 bp, ~220 bp
and ~110 bp for M. obtusa; three bands of ~260 bp, ~130
bp and ~70 bp for M. furfur were obtained (Table-2 and
Fig-2). BstF51 digestion yielded two bands of ~480 bp and
~100 bp for M. globosa; two bands of ~500 bp and ~70 bp
for M. obtusa and M. restricta; two bands of ~400 bp and
~180 bp for M. sympodialis and M. furfur were obtained.

PCR products were evaluated by 1.5% (w/v) agarose gel

electrophoresis in tris-boric acid-EDTA (TBE) buffer
stained with ethidium bromide. The target part of the
26rDNA region was amplified for the Malassezia isolates,
to produce a PCR product approximately 580bp in length .
PCR products were used for further restriction fragment
length polymorphism (RFLP) by CfoI and BstF51 (Sigma-
Aldrich, India) enzymes. Digestion was performed by incu-
bating a 20 L aliquot of PCR products with 10 U of the
enzyme in a final reaction volume of 25 L for 4 hours at
37C and 65. The products were visualized by 2%
agarose gel electrophoresis in a TBE buffer (Fig 1).

RESULTS AND DISCUSSION

Study area was chosen on the basis of convenience to Fig -1: 26S rDNA PCR Products before Digestion with CfoI and BstF51
perform the sampling. The patients were advised not to (Lane 1-100 bp ladder, L2- M. globosa, L3- M. furfur, L4- M. restricta, L5-
wash their scalp preceding a week of sample collection. M. sympodialis, L6- M. obtusa, L7- Reference strain)
Among the 78 individuals selected, 33 were males and 45
were females aged between 20-30 years. Of 78 samples
tested, 50 were reported 10% KOH positive.

Colony morphology

White to cream coloured colonies were observed in

Dixons agar after 3-5 days of incubation at 37C.

Microscopic appearance

Budding yeast like cells revealed Spaghetti and meat ball

appearance were observed under microscope when
stained with Loefflers methylene blue stain. The colony
morphology and microscopic appearance were compared
with the reference strain Malassezia furfur MTCC 1374.

Culture based identification of Malassezia spp.

The isolates were identified by culture based techniques

such as catalase test, tween assimilation, esculin
hydrolysis and glycine assimilation. Biochemical
characterization of positive samples revealed 22 isolates
were M. globosa, 12 were M. furfur, 9 were M. sympodialis,
4 were M. obtusa and 3 were M. restricta (Table-1).

Molecular characterization of the isolates

The 26S rDNA PCR amplified a ~580-bp product. After
Fig -2: 26S rDNA PCR Products after Digestion with CfoI and BstF51
CfoI digestion, two bands of ~480 and ~100 bp were (Table 2-100 bp ladder, L2- M. globosa, L3- M. furfur, L4- M. restricta, L5-
obtained for M. globosa; two bands of ~390 bp and ~190 M. sympodialis, L6- M. obtusa, L7- Reference strain)

Epidemiology and Characterization of lipophilic yeast (Malassezia) from human dandruff samples using cultural and molecular methods
Sharma and Sibi 071

Table- 1: Phenotypic features in the isolates

Isolates Catalase test Tween 20 Tween 40 Tween 60 Tween Esculin Glycine Identification
80 hydrolysis assimilation
I1 + + + + + - + M. furfur
I2 + + + + + - + M. furfur
I3 + - + + + + - M. sympodialis
I4 + - + + + + - M. sympodialis
I5 + + + + + - + M. furfur
I6 + - - - - - - M. globosa
I7 + + + + + - + M. furfur
I8 + + + + + - + M. furfur
I9 + + + + + - + M. furfur
I10 + - + + + + - M. sympodialis
I11 + - - - - + - M. obtusa
I12 + - - - - - - M. restricta
I13 + - - - - - - M. globosa
I14 + - - - - - - M. globosa
I15 + - - - - - - M. globosa
I16 + - - - - - - M. globosa
I17 + + + + + - + M. furfur
I18 + + + + + - + M. furfur
I19 + - + + + + - M. sympodialis
I20 + - - - - - - M. globosa
I21 + + + + + - + M. furfur
I22 + - - - - - - M. globosa
I23 + - - - - - - M. globosa
I24 + - - - - - - M. globosa
I25 + - + + + + - M. sympodialis
I26 + - - - - - - M. globosa
I27 + + + + + - + M. furfur
I28 + - + + + + - M. sympodialis
I29 + - - - - + - M. obtusa
I30 + - - - - + - M. obtusa
I31 + - - - - - - M. globosa
I32 + - + + + + - M. sympodialis
I33 + - - - - - - M. restricta
I34 + - - - - - - M. globosa
I35 + - - - - - - M. globosa
I36 + - - - - - - M. globosa
I37 + - + + + + - M. sympodialis
I38 + - - - - - - M. globosa
I39 + - - - - - - M. globosa
I40 + - - - - - - M. globosa
I41 + + + + + - + M. furfur
I42 + - - - - + - M. obtusa
I43 + - - - - - - M. globosa
I44 + - - - - - - M. globosa
I45 + - + + + + - M. sympodialis
I46 + - - - - - - M. restricta
I47 + + + + + - + M. furfur
I48 + - - - - - - M. globosa
I49 + - - - - - - M. globosa
I50 + - - - - - - M. globosa
Ref + + + + + - + M. furfur MTCC1374
(+) positive; (-) negative

The genus of Malassezia consists of at least fourteen
different species, which have been identified based on
biochemical and morphological features. Macroscopic and
microscopic characteristics and some physiological
aspects allow species level differentiation of the yeasts
(Mayser et al., 1997, Guillot et al., 1996). For example, the
assimilation of Tween 20, 40, 60, and 80 by M. furfur, M.
sympodialis, and M. slooffiae yields a specific pattern that
easily differentiates them from other species. In this work,
thorough characterization of Malassezial isolates with an
atypical Tween 80 assimilation pattern was performed,
and the results support the view that these isolates
correspond to the genus Malassezia. Tween assimilation
is based on the ability of Malassezia spp. to use different

Epidemiology and Characterization of lipophilic yeast (Malassezia) from human dandruff samples using cultural and molecular methods
 Int. Res. J. Biochem. Biotechnol. 072

Table -2: RFLP 26S rDNA PCR products generated by CfoI, and BstF51 respectively, for Malassezia spp

Isolates 26SrDNA amplification product (bp) CfoI digest (bp) BstF51 digest (bp)
M. globosa I16 580 480, 100 480, 100
M. obtusa I11 580 250, 220, 110 500, 70
M. restricta I33 580 NRSa 500, 70
M. sympodialis I4 580 390, 190 400, 180
M. furfur I2 580 260, 130, 70 400, 180
M. furfur MTCC 1374 580 260, 130, 70 400, 180
a
NRS, no restriction site

fatty acids. The atypical assimilation pattern of the isolates Malassezia species (Zhang et al., 2012; Sibi et al., 2014)
involving an inability to utilize Tween 20, 40 and 60, while others report M. sympodialis or M. furfur as the major
reveals a metabolic variation probably resulting from species of pitryiasis versicolor (Yim et al., 2010). The PCR
alternate gene expression. The tween diffusion test products and the products of digestion revealed
allowed the differentiation of most Malassezia species in differences between the isolates with the atypical tween
the study population. Guillot et al., (1996) reported a assimilation pattern and the reference strain M. furfur
method of identification based on lipid usage pattern, MTCC 1374.
catalase reaction, growth temperature and cell shape.
Hammer and Riley (2000) reported the production of a
precipitate by M. sympodialis and M. globosa on Dixon's CONCLUSION
agar. Mayser et al. (1997) reported that
This study proved the variations in the species distribution
some Malassezia species hydrolyzed esculin and
among individuals. To facilitate adequate treatment,
assimilated polyethoxylated castor oil and these properties
accurate identifications of the species are needed to obtain
could be used to differentiate among species (Gueho et
a better understanding of the presence of each individual
al., 1998).
species in the etiology of dandruff. In the selected
individuals, M. globosa was found as predominant species
However, biochemical and morphological features often
followed by M. furfur. The other species identified were M.
do not allow the rapidly and exact characterization of
sympodialis, M. obtusa and M. restricta. Molecular
related Malassezia species, so molecular approaches
approaches are most accurate methods to identify a
need to be used in studies of the epidemiology of the
species and PCR-RFLP was carried out for the
lipophilic yeast (Gueho and Meyer, 1989, Guillot et al.,
differentiation of Malassezia species. Molecular studies
2000). Various molecular methods have been used for
using restriction enzymes revealed the different band
molecular typing of Malassezia species (Guillot and
patterns in the gel electrophoresis. Malassezia furfur
Gueho, 1995; Makimura et al., 2000; Cabanes et al., 2007;
MTCC 1374 was used as reference and different sizes of
Cafarchia et al., 2007 Aizawa et al., 2001; Theelen et al.,
digested products were obtained with varying species. The
2001; Castella et al., 2005; Hossain et al., 2007).
results prove the efficiency of molecular methods to
Molecular methods may resolve the time consuming and
identify a dandruff causative agent among closely related
the difficulties in interpretation of some morphological and
Malassezia species.
physiological patterns, RFLP method was used in this
study which enables to examine genetic variations through
cleaving the amplified DNA with CfoI and BstF51
ACKNOWLEDGMENTS
restrictions enzyme and analyzing the patterns of the
fragments.
Financial assistance in the form of Minor Research Project
(No. MRP(S)-0414/13-14/KABA105/UGC-SWRO) from
Accurate identifications of the species are needed to
University Grants Commission, New Delhi is duly
obtain a better understanding of the role of each individual
acknowledged.
species in the etiology of disease. This study used the
PCR-RFLP method for the identification of the Malassezia
genus in order to determine the specific identification of
REFERENCES
Malassezia species and the diagnosis of related infections.
M. globosa and M. furfur were isolated significantly more Affes M, Ben Salah S, Makni F, Sellami H, Ayadi A. (2009).
often than other species in this study. Hedayati et al., Molecular identification of Malassezia species isolated
(2010) reported 58% M. globosa from patients with from dermatitis affections. Mycoses. 52: 251-256.
seborrheic dermatitis. Zomorodian et al., (2008) reported Aizawa T, Kano R, Nakamura Y, Watanabe S, Hasegawa
that M. furfur is the most common Malassezia species A. (2001). The genetic diversity of clinical isolates of
isolated from psoriasis and healthy individuals. Other Malassezia pachydermatis from dogs and cats. Med.
studies have reported M. globosa as the prevailing Mycol. 39: 329-334.
Epidemiology and Characterization of lipophilic yeast (Malassezia) from human dandruff samples using cultural and molecular methods
Sharma and Sibi 073
Batra R, Boekhout T, Gueho E, Cabanes FJ, Dawson Jr
TL, Gupta AK. (2005). Malassezia Baillon, emerging
clinical yeasts. FEMS Yeast Res. 5: 1101-1113.
Cabanes FJ, Vega S, Castella G. (2010). Malassezia
cuniculi sp. nov., a novel yeast species isolated from
rabbit skin. Med. Mycol. 49: 40-48.
Cabanes FJ, Theelen B, Castella G, Boekhout T. (2007).
Two new lipid dependent Malassezia species from
domestic animals. FEMS Yeast Res. 7: 1064-1076.
Cafarchia C, Otranto D (2004). Association between
phospholipase production by Malassezia pachydermatis
and skin lesions. J. Clin. Microbiol. 42: 4868-4869.
Cafarchia C, Latrofa MS, Testini G, Parisi A, Guillot J,
Gasse, R, Otranto D. (2007). Molecular characterization
of Malassezia isolates from dogs using three distinct
genetic markers in nuclear DNA. Mol. Cell. Probes. 21:
229-238.
Castella G, Hernandez JJ, Cabanes FJ, (2005). Genetic
typing of Malassezia pachydermatis from different
domestic animals. Vet. Microbiol. 108: 291296.
Duarte ER, Hamdan JS, (2009). RAPD differentiation of
Malassezia spp. from cattle, dogs and humans.
Mycoses. 53: 48-56.
Gonzalez A, Sierra R, Cardenas ME, Grajales A, Restrepo
S, Cepero de Garcia MC, Celis A. (2009). Physiological
and molecular characterization of atypical isolates of
Malassezia furfur. J. Clin. Microbiol. 47: 48-53.
Gueho E,Meyer SA. (1989). A reevaluation of the genus
Malassezia by means of genome comparison. Antonie
Van Leeuwenhoek. 55: 245-251.
Gueho E, Boekhout T, Ashbee HR, Guillot J, Van Belkum
A, Faergemann J. (1998). The role of Malassezia
species in the ecology of human skin and as
pathogens. Med. Mycol. 36: 220-229.
Guillot J, Gueho E. (1995). The diversity of Malassezia
yeasts confirmed by rRNA sequence and nuclear DNA
comparisons. Antonie Van Leeuwenhoek 67: 297-314.
Guillot J, Deville M, Berthelemy M, Provost F, Gueho E.
(2000). A single PCR-restriction endonuclease analysis
for rapid identification of Malassezia species. Lett. Appl.
Microbiol. 31:400-403.
Guillot J, Gueho E, Lesourd M, Midgley G, Chevrier G,
Dupont B. (1996). Identification of Malassezia species.
J. Mycol. Med. 6:103-110.
Guillot J, Gueho E. (1995). The diversity of Malassezia
yeasts confirmed by rRNA sequence and nuclear DNA
comparison. Antonie Van Leeuwenhoek. 67: 297-314.
Gupta AK, Bluhm R, Summerbell R. (2002). Pityriasis
versicolor. J. Eur. Acad. Dermatol. Venereol. 16 (1): 19-
33.
Hammer KA, Riley TV. (2000). Precipitate production by
some Malassezia species on Dixon's agar. Med.
Mycol. 38:105-107.
Harada K, Saito M, Sugita T, Tsuboi R. (2015). Malassezia
species and their associated skin diseases. J Dermatol.
42(3): 250-257.
Hedayati MT, Hajheydari Z, Hajjar F, Ehsani A, Shokohi T,
Mohammadpour R. (2010). Identification of Malassezia
Epidemiology and Characterization of lipophilic yeast (Malassezia) from human dandruff samples using cultural and molecular methods
species isolated from Iranian seborrhoeic dermatitis
patients. Eur. Rev. Med. Pharmacol. Sci. 14(1): 63-68.
Hirai A, Kano R, Makimura K, Duarte ER, Hamdan JS,
Lachance MA, et al. (2004). Malassezia nana sp. nov, a
novel lipid-dependent yeast species isolated from
animals. Int. J. Syst. Evol. Microbiol. 54: 623-627.
Hossain H, Landgraf V, Weiss R, Mann M, Hayatpour J,
Chakraborty T, Mayser P, (2007). Genetic and
biochemical characterization of Malassezia
pachydermatis with particular attention to pigment-
producing subgroups. Med. Mycol. 45: 41-49.
Ilahi A, Hadrich I, Neji S, Trabelsi H, Makni F, Ayadi A.
(2017). Real-Time PCR identification of six Malassezia
species.Curr. Microbiol. 74(6): 671-677.
Makimura K, Tamura Y, Kudo M, Uchida K, Saito H,
Yamaguchi H. (2000). Species identification and strain
typing of Malassezia species stock strains and clinical
isolates based on the DNA sequences of nuclear
ribosomal internal transcribed spacer 1 regions. J. Med.
Microbiol. 49: 29-35.
Mayser P, Haze P, Papavassilis C, Pickel M, Gruender K,
Gueho E. (1997). Differentiation of Malassezia species:
selectivity of Cremophor EL, castor oil and ricinoleic acid
for M. furfur. Br. J. Dermatol. 137:208-213.
Midgley G, Gueho E, Guillot J. (1998). Diseases caused
by Malassezia species. In: Ajello J, Hay RJ, eds. Medical
Mycology, Topley and Wilsons Microbiology and
Microbial Infections London, Sidney, Auckland: Arnold,
1998: 201-211.
Park HK, Ha MH, Park SG, Kim MN, Kim BJ, Kim W.
(2012). Characterization of the fungal microbiota
(mycobiome) in healthy and dandruff-afflicted human
scalps. PloS one.7(2): e32847.
Petry V, Tanhausen F, Weiss L, Milan T, Mezzari A, Weber
MB. (2011). Identification of Malassezia yeast species
isolated from patients with pityriasis versicolor. An. Bras.
Dermatol. 86: 803-806.
Prohic A, Jovovic Sadikovic T, Krupalija-Fazlic M,
Kuskunovic-Vlahovljak S. (2016). Malassezia species in
healthy skin and in dermatological conditions. Int. J.
Dermatol. 55: 494-504.
Sambrook J, Fritsch EF, Maniatis T. (Eds). Molecular
cloning: a laboratory manual. 2 nd ed. New York: Cold
Spring Harbor Laboratory 1989. 411p.
Sibi G, Alam MA, . Shah J, Razak M. (2014). Susceptibility
pattern of Malassezia species to selected plant extracts
and antifungal agents. International Journal of Green
Pharmacy. 8(14): 226-230.
Sugita T, Kodama M, Saito M, Ito T, Kato Y, Tsuboi R,
Nishikawa A. (2003). Sequence diversity of the
intergenic spacer region of the rRNA gene of Malassezia
globosa colonizing the skin of patients with atopic
dermatitis and healthy individuals. J. Clin. Microbiol. 41:
3022-3027.
Sugita T, Tajima M, Amaya M, Tsuboi R, Nishikawa A.
(2004). Genotype analysis of Malassezia restricta as the
major cutaneous flora in patients with atopic dermatitis
and healthy subjects. Microbiol. Immunol. 48: 755-759.
 Int. Res. J. Biochem. Biotechnol. 074

Sugita T, Takeo K, Hamas K, Virtudazo E, Takashima M,

Nishikawa A, et al. (2005). DNA sequence diversity of
intergenic spacer 1 region in the non-lipid-dependent
species of Malassezia pachydermatis isolated from
animals. Med. Mycol. 43: 21-26.
Theelen N, Silvestri M, Gueho E, van Belkum A, Boekhout
T, (2001). Identification and typing of Malassezia yeast
using amplified fragment length polymorphism
(AFLPTM), random amplified polymorphic DNA (RAPD)
and denaturing gradient gel electrophoresis (DGGE).
FEMS Yeast Res. 1: 79-86.
Vuran E, Karaarslan A, Karasartova D, Turegun B, Sahin
F. (2014). Identification of Malassezia species from
pityriasis versicolor lesions with a new multiplex PCR
method. Mycopathologia. 177: 41-49.
Yim SM, Kim JY, Ko JH, Lee YW, Choe YB, Ahn KJ.
(2010). Molecular analysis of Malassezia microflora on
the skin of the patients with atopic dermatitis. Ann.
Dermatol. 22(1): 41-47.
Zhang E, Tanaka T, Tsuboi R, Makimura K, Nishikawa A,
Sugita T. (2012). Characterization of Malassezia
microbiota in the human external auditory canal and on
the sole of the foot. Microbiology and immunology.
Micobiol Immunol. 56(4): 238-244.
Zomorodian K, Mirhendi H, Tarazooie B, Zeraati H, Hallaji
Z, Balighi K. (2008). Distribution of Malassezia species
in patients with psoriasis and healthy individuals in
Tehran, Iran. J. Cutan. Pathol. 35(11): 1027-1031.

Accepted 25 July 2017

Citation: Sharma S, Sibi G (2017). Epidemiology and

Characterization of lipophilic yeast (Malassezia) from
human dandruff samples using cultural and molecular
methods. International Research Journal of Biochemistry
and Biotechnology, 4(1): 068-074.

Copyright: 2017 Sharma and Sibi. This is an open-

access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium,
provided the original author and source are cited.

Epidemiology and Characterization of lipophilic yeast (Malassezia) from human dandruff samples using cultural and molecular methods